4. The method of claim 2, wherein said composition comprises nucleic acid
encoding a polypeptide having the amino acid sequence set forth in SEQ ID
NO:2.

5. The method of claim 1, wherein said cancer cells express a mutant
version of a p53 polypeptide, and said composition decreases said USP10
polypeptide expression or activity.

6. The method of claim 5, wherein said composition comprises an
antagonist of USP10 polypeptide mediated stabilization of p53
polypeptides.

7. The method of claim 6, wherein said antagonist comprises nucleic acid
having the ability to induce RNA interference against expression of said
USP10 polypeptide.

8. The method of claim 7, wherein said USP10 polypeptide is a human USP10
polypeptide.

9. A method for treating cancer in a mammal, wherein said method
comprises: (a) identifying a mammal as having cancer cells that express a
reduced level of wild-type p53 polypeptides or that express a mutant p53
polypeptide, (b) administering a USP10 polypeptide or composition that
increases USP10 polypeptide expression or activity within said cancer
cells if said mammal is identified as having cancer cells that express
said reduced level of wild-type p53 polypeptides, and (c) administering a
composition that decreases USP10 polypeptide expression or activity
within said cancer cells if said mammal is identified as having cancer
cells that express said mutant p53 polypeptide.

10. A method for identifying an antagonist of USP10 polypeptide mediated
stabilization of p53 polypeptides, wherein said method comprises
determining if the stabilization level of a ubiquinated p53 polypeptide
contacted with a USP10 polypeptide in the presence of a test agent is
less than the stabilization level of said ubiquinated p53 polypeptide
contacted with said USP10 polypeptide in the absence of said test agent,
wherein the presence of said stabilization level of said ubiquinated p53
polypeptide contacted with said USP10 polypeptide in the presence of said
test agent that is less than said stabilization level of said ubiquinated
p53 polypeptide contacted with said USP10 polypeptide in the absence of
said test agent indicates that said test agent is said antagonist.

11. A method for identifying an agonist of USP10 polypeptide mediated
stabilization of p53 polypeptides, wherein said method comprises
determining if the stabilization level of a ubiquinated p53 polypeptide
contacted with a USP10 polypeptide in the presence of a test agent is
greater than the stabilization level of said ubiquinated p53 polypeptide
contacted with said USP10 polypeptide in the absence of said test agent,
wherein the presence of said stabilization level of said ubiquinated p53
polypeptide contacted with said USP10 polypeptide in the presence of said
test agent that is greater than said stabilization level of said
ubiquinated p53 polypeptide contacted with said USP10 polypeptide in the
absence of said test agent indicates that said test agent is said
agonist.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application
Ser. No. 61/260,637, filed Nov. 12, 2009 and U.S. Provisional Application
Ser. No. 61/241,152, filed Sep. 10, 2009. The disclosures of the prior
applications are considered part of (and are incorporated by reference
in) the disclosure of this application.

BACKGROUND

[0002] 1. Technical Field

[0003] This document relates to methods and materials involved in
modulating deubiquitinases and ubiquitinated polypeptides (e.g., tumor
suppressors such as wild-type p53 polypeptides). For example, this
document relates to methods and materials for increasing or decreasing
deubiquitinase expression or activity, methods and materials for
stabilizing or de-stabilizing ubiquitinated polypeptides, and methods and
materials for treating cancer.

[0004] 2. Background Information

[0005] p53 is a tumor suppressor that is mutated in more than 50% of human
cancers and whose major function is regulating cell fate following
cellular stress and repressing the propagation of damaged cells (Lane,
1992; Riley et al., 2008; Vogelstein et al., 2000). p53 functions as a
transcription factor, and through its target genes regulates a variety of
cellular functions, from cellular senescence, to energy metabolism, DNA
repair, cell differentiation, cell cycle progression and apoptosis. In
addition to the activation of transcription, p53 can also act as a
repressor of transcription, as it does in the suppression of CD44, a
protein implicated in tumorigenesis (Godar et al., 2008). Finally, p53
also has transcription-independent functions, such as regulating
apoptosis through protein-protein interactions (Moll et al., 2005).

[0007] Some cancer cells can express reduced levels of p53 polypeptides,
while other cancer cells can express average or elevated levels of a
mutant version of a p53 polypeptide. As described herein, USP10
polypeptides can interact with and deubiquinate wild-type or mutant p53
polypeptides, thereby increasing their stability. In the cases of cancer
cells having reduced levels of wild-type p53 polypeptides, the methods
and materials provided herein can be used to increase USP10 polypeptide
expression or activity, thereby increasing the stability of the wild-type
p53 polypeptides. This can result in an increased level of wild-type p53
polypeptides within the cancer cells, thereby resulting in reduced cancer
cell proliferation and increased cancer cell apoptosis. In the cases of
cancer cells that express mutant p53 polypeptides, the methods and
materials provided herein can be used to decrease USP10 polypeptide
expression or activity, thereby decreasing the stability of the mutant
p53 polypeptides. This can result in a decreased level of mutant p53
polypeptides within the cancer cells, thereby resulting in reduced cancer
cell proliferation and increased cancer cell apoptosis.

[0008] In general, one aspect of this document features a method for
reducing cancer cell proliferation in a mammal having cancer cells. The
method comprises, or consists essentially of, administering a composition
to the mammal under conditions wherein the composition modulates USP10
polypeptide expression or activity within the cancer cells, thereby
reducing cancer cell proliferation. The cancer cells can have a reduced
level of wild-type p53 polypeptide expression, and the composition can
increase USP10 polypeptide expression or activity. The composition can
comprise nucleic acid encoding a USP10 polypeptide. The composition can
comprise nucleic acid encoding a polypeptide having the amino acid
sequence set forth in SEQ ID NO:2. The cancer cells can express a mutant
version of a p53 polypeptide, and the composition can decrease USP10
polypeptide expression or activity. The composition can comprise an
antagonist of USP10 polypeptide mediated stabilization of p53
polypeptides. The antagonist can comprise nucleic acid having the ability
to induce RNA interference against expression of the USP10 polypeptide.
The USP10 polypeptide can be a human USP10 polypeptide.

[0009] In another aspect, this document features a method for treating
cancer in a mammal. The method comprises, or consists essentially of, (a)
identifying a mammal as having cancer cells that express a reduced level
of wild-type p53 polypeptides or that express a mutant p53 polypeptide,
(b) administering a USP10 polypeptide or a composition that increases
USP10 polypeptide expression or activity within the cancer cells if the
mammal is identified as having cancer cells that express the reduced
level of wild-type p53 polypeptides, and (c) administering a composition
that decreases USP10 polypeptide expression or activity within the cancer
cells if the mammal is identified as having cancer cells that express the
mutant p53 polypeptide.

[0010] In another aspect, this document features a method for identifying
an antagonist of USP10 polypeptide mediated stabilization of p53
polypeptides. The method comprises, or consists essentially of,
determining if the stabilization level of a ubiquinated p53 polypeptide
contacted with a USP10 polypeptide in the presence of a test agent is
less than the stabilization level of the ubiquinated p53 polypeptide
contacted with the USP10 polypeptide in the absence of the test agent,
wherein the presence of the stabilization level of the ubiquinated p53
polypeptide contacted with the USP10 polypeptide in the presence of the
test agent that is less than the stabilization level of the ubiquinated
p53 polypeptide contacted with the USP10 polypeptide in the absence of
the test agent indicates that the test agent is the antagonist.

[0011] In another aspect, this document features a method for identifying
an agonist of USP10 polypeptide mediated stabilization of p53
polypeptides. The method comprises, or consists essentially of,
determining if the stabilization level of a ubiquinated p53 polypeptide
contacted with a USP10 polypeptide in the presence of a test agent is
greater than the stabilization level of the ubiquinated p53 polypeptide
contacted with the USP10 polypeptide in the absence of the test agent,
wherein the presence of the stabilization level of the ubiquinated p53
polypeptide contacted with the USP10 polypeptide in the presence of the
test agent that is greater than the stabilization level of the
ubiquinated p53 polypeptide contacted with the USP10 polypeptide in the
absence of the test agent indicates that the test agent is the agonist.

[0012] In another aspect, this document features a method for assessing
the p53 genotype of a cancer cell. The method comprises, or consists
essentially of, determining the level of USP10 polypeptide expression in
the cancer cell, diagnosing the cancer cell as having wild-type p53 if
the cancer cell contains a reduced level of USP10 polypeptide expression,
and diagnosing the cancer cell as having mutant p53 if the cancer cell
contains an increased level of USP10 polypeptide expression.

[0013] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention pertains. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and not intended
to be limiting.

[0014] Other features and advantages of the invention will be apparent
from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0015] FIG. 1. USP10 interacts with p53. (A and D) U2OS cell lysates were
subjected to immunoprecipitation with control IgG or anti-USP10
antibodies. The immunoprecipitates were then blotted with anti-p53,
anti-Mdm2, or anti-USP10 antibodies. (B and C) HCT116 p53.sup.+/+ and
p53.sup.-/- cell lysates were subjected to immunoprecipitation with
control IgG or anti-USP10 antibodies (B) or anti-p53 antibodies (C). The
immunoprecipitates were then blotted with anti-p53 or anti-USP10
antibodies. (HC: Heavy Chain). (E). Purified FLAG-tagged USP10 was
incubated with GST or GST-p53 coupled to GSH-Sepharose. Proteins retained
on Sepharose were then blotted with indicated antibodies. (F) Constructs
encoding FLAG-tagged full-length (FL) or deletion mutations of USP10 were
transfected into H1299 cells. Forty-eight hours after transfection, cells
were lysed, and cell lysates were incubated with GST or GST-p53 coupled
to GSH-Sepharose. Proteins retained on Sepharose were analyzed with the
indicated antibody.

[0016] FIG. 2. USP10 stabilizes and deubiquitinates p53. (A) HCT116 cells
were transfected with vectors or constructs encoding FLAG-tagged USP10.
Forty-eight hours later, cells were lysed, and cell lysates were blotted
with indicated antibody. (B) HCT116 cells were infected with lentivirus
encoding indicated shRNAs. 72 hours later, cells were lysed, and cell
lysates were blotted with indicated antibody. (C) HCT116 cells were
stably expressing control shRNA, USP10 shRNA, or USP10 shRNA together
with shRNA-resistant USP10. Cells were treated with cycloheximide (0.1
mg/mL) and harvested at the indicated time. The upper panels show
immunoblots of p53 and USP10. β-actin was included as a control.
Lower panel: quantification of the p53 protein levels relative to
β-actin. (D) HCT116 cells were transfected with indicated plasmids.
Forty-eight hours later, cells were lysed, and cell lysates were blotted
with indicated antibody. (E-G) Regulation of p53 ubiquitination levels in
vivo by USP10. H1299 cells transfected with indicated constructs (E) or
stably expressing control or USP10 shRNA (F) were transfected with
FLAG-p53. Forty-eight hours later, cells were treated with MG132 for 4
hours before harvest. p53 was immunoprecipitated with anti-FLAG
antibodies and immunoblotted with anti-p53 antibodies. (G)
Deubiquitination of p53 in vitro by USP10. Ubiquitinated p53 was
incubated with purified USP10 or USP10CA in vitro and then blotted with
anti-p53 antibodies.

[0017] FIG. 3. Regulation of the subcellular localization of p53 by USP10.
(A) Subcellular localization of USP10 and a ubiquitin-specific protease,
HAUSP. U2OS cells were transfected with constructs encoding FLAG-USP10 or
FLAG-HAUSP. Forty-eight hours later, cells were fixed and stained with
indicated antibodies and DAPI. (B) H1299 cells were cotransfected with
indicated constructs. Forty-eight hours later, cells were treated with
MG132, harvested, and fractionated as described herein. Cellular
fractions were then blotted with indicated antibodies. (C, cytoplasmic;
N, nuclear). A cytoplasmic marker protein (GAPDH) and a nuclear marker
protein (Histone3) were used as controls to confirm the quality of
fractionations. (C) H1299 cells were transfected with indicated
constructs. Forty-eight hours later, the cells were treated with MG132,
fixed, and stained with the indicated antibodies and DAPI. (D) U2OS cells
were infected with lentivirus encoding control shRNA or USP10 shRNA. 72
hours later, cells were treated with MG132, fixed, and stained with the
indicated antibodies or DAPI. (C-D) Right panels: Quantification of cells
with different p53 subcellular localization. Nuc: Nucleus only; Cyto+Nuc:
both cytoplasm and nucleus. The data represent the average of three
experiments, and 150 cells were monitored in each experiment.

[0019] FIG. 5. USP10 translocates into the nucleus and regulates p53
activity following DNA damage. (A) HCT116 cells stably expressing control
shRNA or USP10 shRNA were irradiated (10 Gy), and cells were harvested at
the indicated time. Cell lysates were then blotted with the indicated
antibodies. (B) HCT116 cells were left untreated or treated with 10 Gy
radiation. Four hours later cells were stained with anti-USP10 antibody.
(C) HCT116 cells were irradiated (10 Gy) or left untreated. After four
hours, cells were harvested and fractionated as described herein.
Cellular fractions were then blotted with the indicated antibodies. (D)
HCT116 p53.sup.+/+ or p53.sup.-/- cells stably expressing control shRNA
or USP10 shRNA were left untreated or treated with 10 Gy radiation. After
48 hours, apoptotic cells were determined as described herein. Error bar
represents the mean±SEM of triplicate experiments. ** represents
P<0.01 two tailed student's t test. (E) The same cells in (D) were
treated with 10 Gy radiation, then harvested at the indicated time. Cell
cycle progression was examined by FACS.

[0020] FIG. 6. USP10 phosphorylation by ATM regulates USP10 stabilization,
translocation, and p53 activation following DNA damage. (A) HCT116 cells
were irradiated (10 Gy) and harvested at the indicated times. Cell
lysates and mRNA were then extracted and analyzed by Western blot or
RT-PCR, respectively. (B) HCT116 cells were left untreated or irradiated.
Cells were then treated with cycloheximide (0.1 mg/mL) and harvested at
the indicated times. Cell lysates were then blotted with the indicated
antibodies. (C) HCT116 cells were transfected with FLAG-tagged USP10.
Forty-eight hours later, the cells were left untreated or treated with 10
Gy radiation, 40 J/m2 UV, or 20 mM etoposide. After an additional 1
hour, the cells were harvested. Cell lysates were subjected to
immunoprecipitation with anti-FLAG antibody and immunobloted with
phospho-SQ/TQ (pSQ/TQ) antibody. (D) HCT116 cells were transfected with
FLAG-tagged USP10 and pretreated with DMSO, 25 mM Ku55933, or 3 mM
caffeine. After 2 hours of incubation, cells were left untreated or
treated with 10 Gy radiation. The phosphorylation of USP10 was examined
as in (C). (E) ATM.sup.+/+ or ATM.sup.-/- cells were irradiated (10 Gy)
or left untreated. After one hour, the cells were harvested, and cell
lysates were subjected to immunoprecipitation with anti-USP10 antibody
and blotted with pSQ/TQ antibody. (F). ATM.sup.-/+ or ATM.sup.-/+ cells
were left untreated or irradiated (10 Gy) and were harvested at the
indicated time. Cell lysates were then blotted with the indicated
antibodies. (G) HCT116 cells stably expressing USP10 shRNA were
reconstituted with shRNA resistant FLAG-tagged USP10 WT (wild type),
T42A, S337A or 2SA (T42A and S337A double mutation). Cells were left
untreated or irradiated (10 Gy) and harvested at the indicated time. Cell
lysates were then blotted with the indicated antibodies. (H) HCT116 cells
stably expressing USP10 shRNA were reconstituted with shRNA resistant
FLAG-tagged USP10 WT or 2SA. Cells were left untreated or treated with 10
Gy radiation. USP10 phosphorylation was examined by pSQ/TQ antibody. (I)
Cells the same as (H) were irradiated (10 Gy) or left untreated. After
four hours, cells were harvested and fractionated as described herein.
(J) Cells the same as (H) were left untreated or irradiated (10 Gy).
Cells were harvested at the indicated time, and cell lysates were blotted
with the indicated antibodies. (K) Cells the same as (H) were left
untreated or irradiated. Apoptotic cells were determined 48 hours later.

[0022] FIG. 8. (A) H1299 cells stably transfected with control or USP10
shRNA were transfected with GFP-p53. Forty-eight hours later, the cells
were treated with MG132, fixed, and stained with the indicated antibodies
and DAPI. Right panels: Quanitification of cells with different p53
subcellular localization. Nuc: Nucleus only; Cyto+nuc: both cytoplasm and
nucleus. The data represent the average of three experiments, and 150
cells were monitored in each experiment. (B) Cells the same as FIG. 6H
were left untreated or treated with 10 Gy radiation. After four hours,
the cells were fixed and stained with anti-FLAG antibody or DAPI. Right
panels: Quantification of cells with different USP10 subcellular
localization. Cyto: cytoplasm only; Cyto+Nuc: cytoplasm and nucleus. The
data represent the average of three experiments, and 150 cells were
monitored in each experiment. (C) Cells the same as FIG. 7D-E were lysed,
and cell lysates were blotted with the indicated antibodies.

[0023]FIG. 9 is a listing of a nucleic acid sequence (SEQ ID NO:1) that
encodes a human USP10 polypeptide.

[0024]FIG. 10 is a listing of an amino acid sequence of a human USP10
polypeptide (SEQ ID NO:2).

[0031]FIG. 17A is a concentration curve graph plotting the level of
deubiquitination of Ub-AMC observed following incubation with the
indicated amounts of USP10 polypeptide (μg). FIG. 17B is a graph
plotting the level of deubiquitination of Ub-AMC in the presence (right)
or absence (left) of 4 μg/mL of USP10 polypeptides and the indicated
compound.

[0032]FIG. 18 is a photograph of an immunoprecipitation of HCT116 cell
lysates with an anti-USP10 polypeptide antibody (or control antibody,
IgG) and immunoblotted with anti-G3BP1 polypeptide antibodies or
anti-USP10 polypeptide antibodies.

[0033]FIG. 19A is a schematic diagram of USP10 polypeptides (e.g., full
length and fragments of full length USP10 polypeptides) with a table
indicating that both G3BP1 polypeptides and p53 polypeptides interact
with the N-terminal region (e.g., 1-100 amino acids) of USP10
polypeptides. FIG. 19B is a photograph of an immunoprecipitation of
HCT116 cell lysates with an anti-p53 polypeptide antibody (or control
antibody, IgG) and immunoblotted with anti-USP10 polypeptide antibodies,
anti-p53 polypeptide antibodies, or anti-G3BP1 polypeptide antibodies.
The HCT116 cell lysates were obtained from cells treated with MG132 and
either control shRNA (Ctrl) or shRNA designed to reduce G3BP1 polypeptide
expression (G3BP1). FIG. 19c is a photograph of an immunoprecipitation of
HCT116 cell lysates with an anti-p53 polypeptide antibody (or control
antibody, IgG) and immunoblotted with anti-USP10 polypeptide antibodies,
anti-p53 polypeptide antibodies, or anti-FLAG antibodies. The HCT116 cell
lysates were obtained from cells treated with MG132 and either a control
vector (Vector) or a vector designed to overexpress G3BP1 polypeptides
(FLAG-G3BP1).

[0034]FIG. 20 is a photograph of HCT116 cell lysates immunoblotted with
anti-FLAG antibodies, anti-USP10 polypeptide antibodies, anti-p53
polypeptide antibodies, or anti-β-actin antibodies. The HCT116 cell
lysates were obtained from cells stably transfected with either a control
construct (Ctrl) or an shRNA construct designed to reduce USP10
polypeptide expression (USP10) that were transfected with an empty vector
(Vector), a vector designed to over-express G3BP1 polypeptides
(FLAG-G3BP1), or a vector designed to over-express G3BP2 polypeptides
(FLAG-G3BP2).

[0035]FIG. 21 is a graph plotting cell growth (fold of growth, set cell
number at day 1 as 1) versus time (days) for HCT116 cells stably
expressing either control or an shRNA construct designed to reduce
expression of USP10 polypeptides (USP10shRNA) and transfected with a
control vector (Vector) or a FLAG-G3BP1 construct (G3BP1).

[0036]FIG. 22 is a photograph of an immunoprecipitation of HCT116 cell
lysates with an anti-USP10 polypeptide antibody (or control antibody,
IgG) and immunoblotted with anti-G3BP1 polypeptide antibodies and
anti-USP10 polypeptide antibodies. The HCT116 cell lysates were obtained
from untreated cells or cells treated with 10 Gy irradiation.

[0038] In one embodiment, this document provides methods and materials
related to treating mammals (e.g., humans) having cancer. Examples of
mammals that can be treated as described herein include, without
limitation, humans, monkeys, dogs, cats, cows, horses, pigs, rats, and
mice. Examples of cancers that can be treated as described herein
include, without limitation, renal cancers (e.g., renal cell carcinomas),
pancreatic cancers, breast cancers, and glioma. A mammal can be
identified as having cancer using any appropriate cancer diagnostic
techniques. In some cases, a cancer can be assessed to determine if the
cancer is a cancer with a reduced level of p53 polypeptides (e.g.,
wild-type p53 polypeptides). Any appropriate method can be used to assess
the level of p53 polypeptides within cancer cells. For example, nucleic
acid detection techniques such as RT-PCR or microarray assays can be used
to assess the level of p53 mRNA within cancer cells or polypeptide
detection techniques such as immunohistochemistry or ELISAs can be used
to assess the level of p53 polypeptides within cancer cells.

[0039] As described herein, cancer having a reduced level of wild-type p53
polypeptides can be treated by increasing the level of USP10 polypeptide
expression or activity. The increased level of USP10 polypeptide
expression or activity can stabilize wild-type p53 polypeptides within
the cancer cells, thereby resulting in reduced cancer cell proliferation
and increased cancer cell apoptosis. In some cases, the level of USP10
polypeptide within cancer cells can be increased by administering a
composition containing USP10 polypeptides. In some cases, the level of
USP10 polypeptide expression or activity within cancer cells can be
increased by administering a USP10 polypeptide agonist or a nucleic acid
encoding a USP10 polypeptide to the cancer cells. Such a nucleic acid can
encode a full-length USP10 polypeptide such as a human USP10 polypeptide
having the amino acid sequence set forth in SEQ ID NO:2, or a
biologically active fragment of a USP10 polypeptide having amino acid
residues 520 to 793 of the sequence set forth in SEQ ID NO:2. A nucleic
acid encoding a USP10 polypeptide or fragment thereof can be administered
to a mammal using any appropriate method. For example, a nucleic acid can
be administered to a mammal using a vector such as a viral vector.

[0040] Vectors for administering nucleic acids (e.g., a nucleic acid
encoding a USP10 polypeptide or fragment thereof) to a mammal are known
in the art and can be prepared using standard materials (e.g., packaging
cell lines, helper viruses, and vector constructs). See, for example,
Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey
R. Morgan, Humana Press, Totowa, N.J. (2002) and Viral Vectors for Gene
Therapy: Methods and Protocols, edited by Curtis A. Machida, Humana
Press, Totowa, N.J. (2003). Virus-based nucleic acid delivery vectors are
typically derived from animal viruses, such as adenoviruses,
adeno-associated viruses, retroviruses, lentiviruses, vaccinia viruses,
herpes viruses, and papilloma viruses. Lentiviruses are a genus of
retroviruses that can be used to infect cells (e.g., cancer cells).
Adenoviruses contain a linear double-stranded DNA genome that can be
engineered to inactivate the ability of the virus to replicate in the
normal lytic life cycle. Adenoviruses and adeno-associated viruses can be
used to infect cancer cells.

[0041] Vectors for nucleic acid delivery can be genetically modified such
that the pathogenicity of the virus is altered or removed. The genome of
a virus can be modified to increase infectivity and/or to accommodate
packaging of a nucleic acid, such as a nucleic acid encoding a USP10
polypeptide or fragment thereof. A viral vector can be
replication-competent or replication-defective, and can contain fewer
viral genes than a corresponding wild-type virus or no viral genes at
all.

[0042] In addition to nucleic acid encoding a USP10 polypeptide or
fragment thereof, a viral vector can contain regulatory elements operably
linked to a nucleic acid encoding a USP10 polypeptide or fragment
thereof. Such regulatory elements can include promoter sequences,
enhancer sequences, response elements, signal peptides, internal ribosome
entry sequences, polyadenylation signals, terminators, or inducible
elements that modulate expression (e.g., transcription or translation) of
a nucleic acid. The choice of element(s) that may be included in a viral
vector depends on several factors, including, without limitation,
inducibility, targeting, and the level of expression desired. For
example, a promoter can be included in a viral vector to facilitate
transcription of a nucleic acid encoding a USP10 polypeptide or fragment
thereof. A promoter can be constitutive or inducible (e.g., in the
presence of tetracycline), and can affect the expression of a nucleic
acid encoding a USP10 polypeptide or fragment thereof in a general or
tissue-specific manner. Tissue-specific promoters include, without
limitation, enolase promoter, prion protein (PrP) promoter, and tyrosine
hydroxylase promoter.

[0043] As used herein, "operably linked" refers to positioning of a
regulatory element in a vector relative to a nucleic acid in such a way
as to permit or facilitate expression of the encoded polypeptide. For
example, a viral vector can contain a neuronal-specific enolase promoter
and a nucleic acid encoding a USP10 polypeptide or fragment thereof. In
this case, the enolase promoter is operably linked to a nucleic acid
encoding a USP10 polypeptide or fragment thereof such that it drives
transcription in neuronal tumor cells.

[0044] A nucleic acid encoding a USP10 polypeptide or fragment thereof
also can be administered to cancer cells using non-viral vectors. Methods
of using non-viral vectors for nucleic acid delivery are known to those
of ordinary skill in the art. See, for example, Gene Therapy Protocols
(Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana
Press, Totowa, N.J. (2002). For example, a nucleic acid encoding a USP10
polypeptide or fragment thereof can be administered to a mammal by direct
injection (e.g., an intratumoral injection) of nucleic acid molecules
(e.g., plasmids) comprising nucleic acid encoding a USP10 polypeptide or
fragment thereof, or by administering nucleic acid molecules complexed
with lipids, polymers, or nanospheres.

[0045] A nucleic acid encoding a USP10 polypeptide or fragment thereof can
be produced by standard techniques, including, without limitation, common
molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid
synthesis techniques, and combinations of such techniques. For example
PCR or RT-PCR can be used with oligonucleotide primers designed to
amplify nucleic acid (e.g., genomic DNA or RNA) encoding a USP10
polypeptide or fragment thereof.

[0046] In some cases, a nucleic acid encoding a USP10 polypeptide or
fragment thereof can be isolated from a healthy mammal or a mammal having
cancer. For example, a nucleic acid that encodes a wild type USP10
polypeptide having the amino acid sequence set forth in SEQ ID NO:2 can
be isolated from a human containing that nucleic acid. The isolated
nucleic acid can then be used to generate a viral vector, for example,
which can be administered to a mammal so that the level of a USP10
polypeptide or fragment thereof in cancer cells within the mammal is
increased.

[0047] In some cases, a cancer can be assessed to determine if the cancer
is a cancer that expresses a mutant version of a p53 polypeptide.
Examples of mutant p53 polypeptide include, without limitation, those
having the amino acid sequence as set forth elsewhere ("The UMD-p53
database: New mutations and analysis tools," Christophe Beroud and
Thierry Soussi, Human Mutation, Volume 21:p. 176-181; and Berglind et
al., Cancer Biol. Ther., 7(5):699-708 (2008)). Any appropriate method can
be used to assess cancer cells for a mutant version of a p53 polypeptide.
For example, nucleic acid detection techniques such as RT-PCR or
microarray assays can be used to assess cancer cells for a mutant version
of a p53 polypeptide or polypeptide detection techniques such as
immunohistochemistry or ELISAs can be used to assess cancer cells for a
mutant version of a p53 polypeptide.

[0048] As described herein, cancers that express a mutant version of a p53
polypeptide can be treated by decreasing the level of USP10 polypeptide
expression or activity. The decreased level of USP10 polypeptide
expression or activity can destabilize mutant p53 polypeptides within the
cancer cells, thereby resulting in reduced cancer cell proliferation and
increased cancer cell apoptosis. In some cases, the level of USP10
polypeptide expression or activity within cancer cells can be decreased
by administering a USP10 polypeptide antagonist to the cancer cells.
Examples of USP10 polypeptide antagonists that can have the ability to
decrease or inhibit the level of USP10 polypeptide activity within a cell
include, without limitation, N-ethylmaleimide, Z-phe-ala fluoromethyl
ketone, chymostatin, E-64 (trans-Epoxysuccinyl-L-leucylamido
(4-guanidino)butane, E-64d ((2S,
3S)-trans-Epoxysuccinyl-L-leuclamido-3-methylbutane ethyl ester),
antipain dihydrochloride, cystatin, and cyano-indenopyrazine derivatives.
In some cases, a USP10 polypeptide antagonist can be a nucleic acid
molecule designed to induce RNA interference (e.g., an RNAi molecule or a
shRNA molecule). Examples of such shRNA molecules include, without
limitation, those set forth in FIG. 11. Nucleic acid molecules designed
to induce RNA interference against USP10 polypeptide expression can be
administered to a mammal using any appropriate method including, without
limitation, those methods described herein. For example, a nucleic acid
designed to induce RNA interference against USP10 polypeptide expression
can be administered to a mammal using a vector such as a viral vector.

[0050] In some cases, an USP10 polypeptide antagonist can be a
non-polypeptide molecule (e.g., a nucleic acid-based molecule such as an
shRNA or RNAi molecule). In some cases, an USP10 polypeptide antagonist
can be a non-G3BP1 polypeptide molecule (e.g., a nucleic acid-based
molecule such as an shRNA or RNAi molecule).

[0051] This document also provides methods and materials related to
identifying agonists or antagonists of USP10 polypeptide mediated
stabilization of p53 polypeptides. For example, this document provides
methods and materials for using USP10 polypeptides and p53 polypeptides
(e.g., ubiquinated p53 polypeptides) to identify agents that increase or
decrease the ability of the USP10 polypeptides to stabilize the p53
polypeptides. In some cases, the stability of ubiquinated p53
polypeptides treated with USP10 polypeptides in the presence and absence
of a test agent can be assessed to determine whether or not the test
agent increases or decreases the stability of the ubiquinated p53
polypeptides. An agent that increases the stability of the ubiquinated
p53 polypeptides in a manner dependent on the USP10 polypeptide can be an
agonist of USP10 polypeptide mediated stabilization of p53 polypeptides,
and an agent that decreases the stability of the ubiquinated p53
polypeptides in a manner dependent on the USP10 polypeptide can be an
antagonist of USP10 polypeptide mediated stabilization of p53
polypeptides. The stability of ubiquinated p53 polypeptides can be
assessed using polypeptide assays capable of detecting intact full-length
polypeptide or degraded polypeptides. USP10 polypeptide agonists and
antagonists can be identified by screening test agents (e.g., from
synthetic compound libraries and/or natural product libraries). Test
agents can be obtained from any commercial source and can be chemically
synthesized using methods that are known to those of skill in the art.
Test agents can be screened and characterized using in vitro cell-based
assays, cell free assays, and/or in vivo animal models.

[0052] USP10 agonists or antagonists can be identified using an in vitro
screen that includes using purified His-tagged USP10 polypeptide together
with ubiquitin-AMC (BIOMOL) as the substrate. Ubiquitin-AMC is a
fluorogenic substrate for a wide range of deubiquitinylating enzymes
(Dang et al., Biochemistry, 37:1868 (1998)). This fluorescence can allow
high-throughput screen of USP10 agonists and antagonists in vitro.

[0053] In some cases, the expression level of USP10 polypeptides can be
used to assess the p53 genotype of a cancer cell. For example,
identification of cancer cells having an increased level of USP10
polypeptide expression can indicate that the cancer cells contain mutant
p53, while identification of cancer cells having a decreased level of
USP10 polypeptide expression can indicate that the cancer cells contain
wild-type p53.

[0054] The invention will be further described in the following examples,
which do not limit the scope of the invention described in the claims.

[0061] Cycloheximide was purchased from Sigma. For protein turnover
analysis, cycloheximide was added to cell culture medium at the final
concentration of 0.1 mg/mL, and cells were harvested at the indicated
time points. Cells were then lysed, and cell lysates were resolved by
SDS-PAGE and analyzed by Western blot.

Ubiquitination of p53 In Vivo and In Vitro

[0062] The ubiquitination levels of p53 were detected essentially as
described elsewhere (Li et al., Nature, 416:648-653 (2002)). For the in
vivo deubiquitination assay, H1299 cells were transfected with FLAG-p53
or in combination with different expression vectors as indicated. After
48 hours, cells were treated for 4 hour with a proteasome inhibitor MG132
(50 μM) before being harvested. The cell extracts were subjected to
immunoprecipitation with anti-FLAG antibody and blotted with anti-p53
antibodies.

[0063] For the preparation of a large amount of ubiquitinated p53 as the
substrate for the deubiquitination assay in vitro, HEK293 cells were
transfected together with the FLAG-p53, pCMV-Mdm2, and HA-UB expression
vectors. After treatment as described above, ubiquitinated p53 was
purified from the cell extracts with anti-FLAG-affinity column in
FLAG-lysis buffer (50 mM Tris-HCl pH 7.8, 137 mM NaCl, 10 mM NaF, 1 mM
EDTA, 1% Triton X-100, 0.2% Sarkosyl, 1 mM DTT, 10% glycerol and fresh
proteinase inhibitors). After extensive washing with the FLAG-lysis
buffer, the proteins were eluted with FLAG-peptides (Sigma). The
recombinant His-USP10 and USP10CA were expressed in BL21 cells and
purified on the His-tag purification column (Novagen). For the
deubiquitination assay in vitro, ubiquitinated p53 protein was incubated
with recombinant USP10 in a deubiquitination buffer (50 mM Tris-HCl pH
8.0, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol) for 2 hours at
37° C.

Cell Fractionation

[0064] H1299 cells were transfected with the indicated constructs.
Forty-eight hours later, cells were treated for 4 hours with a proteasome
inhibitor, MG132 (50 μM) before being harvested. Cytoplasmic and
unclear fractions were separated by using Paris Kit (Ambion).

Immunofluorescence

[0065] For the p53 translocation assay, H1299 cells were plated on glass
coverslips and transfected with the indicated plasmid. Forty-eight hours
after transfection, 50 μM of proteasome inhibitors (MG132) was added
for 4 hours before fixation. Cells were then fixed in 4% paraformaldehyde
for 10 minutes at room temperature and stained using standard protocols.

Luciferase Assay

[0066] HCT116 p53.sup.-/+ and HCT116 p53.sup.-/- cells were seeded at
8×104 cells/well on 24-well plates. The next day, cells were
transfected with 200 ng of p21 reporter construct and other indicated
plasmids. pRL-TK (50 ng) was included as an internal control. Luciferase
assays were carried out according to the manufacturer's instructions
(Dual-Luciferase Reporter Assay System; Promega). Results were normalized
for expression of pRL-TK as measured by Renilla luciferase activity.

[0068] The soft agar colony-formation assay was performed as described
elsewhere (Shim et al., Proc. Natl. Acad. Sci. USA, 94:6658-6663 (1997)).
Briefly, cells were infected with lentivirus encoding control,
USP10shRNA, or USP10shRNA together with FLAG-tagged USP10. Cells were
then plated in 0.3% top agarose in 35 mm dishes and cultured for two
weeks. Colonies were counted at room temperature under a light microscope
(ECLIPSE 80i; Nikon) using a 4× NA 0.10 objective lens (Nikon).
Images were captured with a camera (SPOT 2 Megasample; Diagnostic
Instruments) and processed using SPOT 4.6 software (Diagnostic
Instruments). Adobe Photoshop and Illustrator were used to generate
figures.

Apoptosis Assay

[0069] Cells were washed with PBS and fixed in 4% paraformaldehyde at room
temperature for 15 minutes. For DAPI staining, cells were stained with 50
μg/mL DAPI. The number of apoptotic cells with nuclear morphology
typical of apoptosis was scored in at least 400 cells in each sample by
using fluorescence microscopy. The reader was blinded to the actual
groups in the fluorescence microscopy.

Tissue Microarray

[0070] The tissue array of kidney cancer samples was purchased from US
Biomax (KD 2083, KD991t, KD804, KD241, KD208t) Immunohistochemical
staining against USP10 (dilution 1:500) was carried out with a IHC
Select® HRP/DAB kit (Cat. DAB50, Millipore). The degree of
immunostainining was determined by board certified pathologists using a
four-tier grading system (0=negative, 1=weak, 2=moderate, and 3=strong
staining intensity) in a blinded manner.

Results

[0071] USP10 Interacts with p53 and Stabilizes p53

[0072] As shown in FIG. 1A-B, USP10 coimmunoprecipitated with p53 in U2OS
and HCT116 p53.sup.-/+ cells, but not HCT116 p53.sup.-/- cells.
Reciprocal immunoprecipitation with anti-p53 also brought down USP10 in
HCT116 p53.sup.+/+, but not in HCT116 p53.sup.-/- cells (FIG. 1c). Unlike
HAUSP, USP10 did not interact with Mdm2 (FIG. 1D). These results suggest
a specific interaction between USP10 and p53 in vivo. However, it is not
clear whether the USP10-p53 interaction is direct. To test this,
recombinant USP10 and p53 were generated and purified. Purified USP10 was
able to interact with p53 under cell-free conditions, suggesting a direct
interaction between USP10 and p53 (FIG. 1E). Further mapping of the
USP10-p53 interaction revealed that the N-terminal region (AA1-AA101),
but not the enzymatic domain of USP10, is required for the interaction
between USP10 and p53 (FIG. 1F).

[0073] USP10 was overexpressed in cells to determine if USP10 could
function to stabilize p53. As shown in FIG. 2A, overexpression of USP10
significantly increased the levels of endogenous p53 and the p53 target
p21. To confirm these results, USP10 expression was knocked-down using
USP10 specific shRNA. The downregulation of USP10 decreased p53 and p21
levels (FIG. 2B). A second USP10 shRNA also exhibited similar effects
(FIG. 2B). These results indicate that USP10 can upregulate p53 levels,
most likely by deubiquitinating and consequently stabilizing p53. To
further confirm that USP10 affects p53 stability, control cells or cells
stably expressing USP10 shRNA were treated with cycloheximide (CHX), and
p53 stability was examined. p53 stability was decreased in cells stably
expressing USP10 shRNA, while reconstitution with shRNA-resistant USP10
restored p53 stability (FIG. 2c). These results demonstrate that USP10
stabilizes p53 in cells.

USP10 Deubiquitinates p53

[0074] USP10 may function to deubiquitinate p53 to counteract the action
of E3 ubiquitin ligases such as Mdm2. Indeed, as shown in FIG. 2D,
although overexpression of Mdm2 significantly induced the degradation of
p53, coexpression of USP10 effectively rescued p53 from Mdm2-induced
degradation. Whether USP10 regulates the levels of p53 ubiquitination in
cells was also examined. As shown in FIG. 2E, Mdm2 induced the
ubiquitination of p53; however, p53 ubiquitination was significantly
diminished by coexpression of USP10. On the other hand, coexpression of
USP10-C488A (USP10CA), a catalytic-inactive USP10 mutant containing a
mutation at the core enzymatic domain (Soncini et al., Oncogene,
20:3869-3879 (2001)), lost the ability to reverse p53 ubiquitination
induced by Mdm2 (FIG. 2E). Conversely, downregulation of USP10 increased
p53 ubiquitination (FIG. 2F). These results indicate that USP10
negatively regulates p53 ubiquitination induced by Mdm2 in cells.
However, from this data alone it is not clear whether USP10's effect on
p53 is direct, since it is possible that USP10 affects another protein,
which in turn affects p53 ubiquitination. To directly examine the
deubiquitination activity of USP10 toward p53, it was determined whether
USP10 could deubiquitinate p53 in a cell free system. USP10 and USP10CA
were purified from bacteria, and ubiquitinated p53 was purified from
cells expressing FLAG-p53, pCMV-Mdm2, and HA-ub. USP10 and ubiquitinated
p53 were then incubated in a cell-free system. As shown in FIG. 2G,
purified wild-type USP10, but not the catalytically inactive USP10CA,
effectively deubiquitinated p53 in vitro. These results demonstrate that
USP10 deubiquitinates p53 both in vitro and in vivo.

USP10 Localizes in the Cytoplasm and Counteracts Mdm2 Action

[0075] Previous studies suggest that ubiquitination of p53 by Mdm2 could
induce p53 translocation from nucleus to cytoplasm (Boyd et al., Nat.
Cell. Biol., 2:563-568 (2000); Geyer et al., Nat. Cell. Biol., 2:569-573
(2000); Li et al., Science, 302:1972-1975 (2003); and Stommel et al.,
Embo J., 18:1660-1672 (1999)). In addition, the cytoplasmic ubiquitin
ligase Parc can ubiquitinate p53 and trap p53 in the cytoplasm (Nikolaev
et al., Cell, 112:29-40 (2003). However, it is not clear whether the
cytoplasmic p53 can be deubiquitinated and returned to the nucleus, since
HAUSP is mainly localized in the nucleus and no cytoplasmic
ubiquitin-specific protease against p53 has been identified. Unlike
HAUSP, USP10 is predominantly localized to the cytoplasm (FIG. 3A). This
result suggests that USP10 is the cytoplasmic deubiquitinase for p53.
Thus, it is possible that USP10 could reverse Mdm2-induced nuclear export
of p53. To test this, cell fractionation experiments were performed.
Expression of Mdm2 was found to induce ubiquitination and nuclear export
of p53, which was reversed by USP10 coexpression (FIG. 3B). To confirm
this result, immunofluorescence assays were performed to detect the
subcellular localization of p53. When H1299 cells were transfected with
GFP-tagged p53, GFP-p53 was readily detected in the nucleus. As
previously demonstrated, when cells were cotransfected with Mdm2, Mdm2
induced cytoplasmic translocation of p53 (Boyd et al., Nat. Cell. Biol.,
2:563-568 (2000); Geyer et al., Nat. Cell. Biol., 2:569-573 (2000); Li et
al., Science, 302:1972-1975 (2003); and Stommel et al., Embo J.,
18:1660-1672 (1999)). However, coexpression of wild-type USP10, but not
catalytically inactive USP10 (USP10CA), reversed Mdm2-induced cytoplasmic
translocation of p53 (FIG. 3C). These results demonstrate that USP10
counteracts Mdm2 by deubiquitinating p53 and inducing p53 translocation
from the cytoplasm back to the nucleus. Therefore, a balance between
USP10 and Mdm2 could determine p53 localization. If so, downregulation of
USP10 could have a similar effect as Mdm2 overexpression. Consistent with
this finding, downregulation of USP10 itself induced nuclear export of
endogenous p53 (FIG. 3D). Similar results were obtained using GFP-p53
(FIG. 8A). These results support a role of USP10 in regulating
homeostasis of p53 in cells.

USP10 is Upregulated and Translocates to the Nucleus Following DNA Damage
and Regulates p53-Dependent DNA Damage Response

[0077] The results provided herein reveal that USP10 can regulate p53
homeostasis in unstressed cells. Since p53 plays a role in DNA damage
response and becomes stabilized following DNA damage, it was examined
whether USP10 is involved in p53 stabilization after DNA damage.
Interestingly, downregulation of USP10 significantly decreased p53
stabilization and the expression of p53 target genes p21 and Bax after
DNA damage (FIG. 5A), suggesting that USP10 also regulates p53
stabilization after DNA damage. Furthermore, it was observed that the
expression of USP10 itself was increased after DNA damage. These results
can be rather surprising, since most DNA damage signaling is thought to
occur in the nucleus. How does USP10, which is located in the cytoplasm,
affect p53 stabilization during DNA damage response? It is possible that
p53 is still actively exported out of the nucleus and gets degraded in
the cytoplasm during DNA damage response, although there is lack of
evidence to support this. Alternatively, USP10 could translocate into the
nucleus to participate in DNA damage response. Indeed, USP10 also
localized in the nucleus following DNA damage as determined by
immunofluorescence (FIG. 5B). To confirm the translocation of USP10, cell
fractionation assays were performed. As shown in FIG. 5c, increased
amounts of USP10 were detected in the nucleus following DNA damage,
confirming a DNA damage-induced translocation of USP10 into the nucleus.

[0078] Since USP10 regulates p53 stabilization following DNA damage,
whether USP10 is required for p53-dependent function during DNA damage
response was examined. As shown in FIG. 5D, downregulation of USP10
inhibited IR-induced apoptosis in HCT116 p53.sup.-/+ cells. The
IR-induced apoptosis in HCT116 p53.sup.-/- cells was blunted, however,
downregulation of USP10 did not have a further effect. Furthermore,
knockdown of USP10 in HCT116 p53.sup.+/+ cells resulted in defective DNA
damage-induced G1 arrest (FIG. 5E). These results are consistent with
decreased Bax and p21 expression in cells with USP10 downregulation (FIG.
5A), and suggest that USP10 is required for p53 activation following DNA
damage.

USP10 Phosphorylation by ATM is Required for its Stabilization and
Translocation Following DNA Damage

[0079] The following experiments were performed to determine the molecular
mechanisms that regulate USP10 upregulation and translocation. Initial
experiments indicated that unlike p21, the upregulation of USP10 occurred
without any change in USP10 mRNA (FIG. 6A), suggesting it is not
regulated at the transcriptional level, and might be regulated at the
posttranslational levels. To examine whether USP10 polypeptides become
stabilized, cells were irradiated, and cells were treated with
cycloheximide. As shown in FIG. 6B, USP10 became more stable in
irradiated cells, suggesting USP10 accumulation after DNA damage is due
to increased stability.

[0080] Phosphorylation is a major posttranslational modification of the
DNA damage response pathway, and it has been shown to enhance protein
stability and activity. For example, p53 is phosphorylated at Ser20 by
the checkpoint kinase Chk2 after IR, which results in p53's dissociation
from Mdm2 and its subsequent stabilization (Chehab et al., Genes Dev.,
14:278-288 (2000); Hirao et al., Science, 287:1824-1827 (2000); and Shieh
et al., Genes Dev., 14:289-300 (2000)). ATM can also directly
phosphorylate p53 at Ser15, so regulating p53 transcriptional activity
and localization (Canman et al., Science, 281:1677-1679 (1998); Siliciano
et al., Genes Dev., 11:3471-3481 (1997); and Zhang and Xiong, Science,
292:1910-1915 (2001)). Therefore, it was examined whether USP10 is
phosphorylated following DNA damage, which might be responsible for its
stabilization and localization. As shown in FIG. 6C, following IR, UV, or
etoposide treatment, USP10 became phosphorylated at SQ/TQ motifs (USP10
polypeptide levels were equalized to specifically examine USP10
phosphorylation in experiments of FIG. 6C-E). The SQ/TQ motifs are
consensus phosphorylation sites for PI3-kinase like kinases (PIKKS), such
as ATM, ATR, and DNA-PK (Abraham, Genes Dev., 15:2177-2196 (2001)), the
major upstream kinases of the DNA damage response pathway. Experiments
were performed to determine whether PIKKs are required for USP10
phosphorylation using the pan-PIKK inhibitor caffeine (Sarkaria et al.,
Cancer Res., 59:4375-4382 (1999)). As shown in FIG. 6D, caffeine
inhibited USP10 phosphorylation after DNA damage. In addition, a specific
ATM inhibitor KU55933 (Hickson et al., Cancer Res., 64:9152-9159 (2004))
also inhibited USP10 phosphorylation after IR. These results suggest that
PIKKS, likely ATM, regulate USP10 phosphorylation after DNA damage. The
role of ATM in USP10 phosphorylation was further confirmed using
ATM.sup.+/+ or ATM.sup.-/- cells. As shown in FIG. 6E, USP10 failed to be
phosphorylated at the SQ/TQ motifs in ATM.sup.-/- cells. Furthermore,
USP10 levels did not increase following DNA damage in ATM.sup.-/- cells
(FIG. 6F). These results indicate that USP10 is phosphorylated by ATM
following DNA damage, which might contribute to its stabilization.

[0081] Experiments were performed to determine the ATM phosphorylation
sites of USP10. ATM specifically phosphorylates SQ/TQ motifs, of which
there are two candidate sites in USP10: T42Q and S337Q. Mutation at
either T42 or 5337 partially affects USP10 stabilization, and mutating
both T42 and S337 (USP10 2SA) abolished USP10 stabilization following DNA
damage (FIG. 6G). Mutation of both T42 and S337 (USP10 2SA) also
abolished USP10 phosphorylation by ATM (FIG. 6H). In addition, the USP10
2SA mutant failed to translocate into the nucleus following DNA damage
(FIG. 6I and FIG. 8B). These results indicate that ATM-mediated
phosphorylation of USP10 is required for USP10 translocation and
stabilization.

[0082] The functional significance of USP10 phosphorylation by ATM was
examined. HCT116 cells stably expressing USP10 shRNA were reconstituted
with shRNA-resistant wild-type USP10 or USP10 2SA. As shown in FIG. 6J,
cells expressing the USP10 2SA mutant exhibited defective p53
stabilization and poor induction of Bax and p21 following DNA damage. In
addition, reconstitution with wild-type USP10, but not the USP10 2SA
mutant, restored DNA damage-induced apoptosis (FIG. 6K). These results
establish the role of USP10 phosphorylation in p53 activation following
DNA damage.

USP10 is Downregulated in Renal Cell Carcinoma

[0083] Since p53 is a tumor suppressor that regulates cell proliferation
and USP10 potentiates p53 function by deubiquitinating p53, it is
possible that USP10 also acts as a tumor suppressor. The results shown in
FIGS. 4D and E demonstrate USP10's ability to inhibit cancer cell
proliferation and lend support to the hypothesis that USP10 functions as
a tumor suppressor in vivo. To further test this hypothesis, the
expression of USP10 in a panel of renal cell carcinoma (RCC) cell lines
was examined. RCC was selected to study USP10 expression because a very
low percentage of RCC cases has been found to have p53 mutations ((Soussi
et al., Hum. Mutat., 15:105-113 (2000)). See, also, the p53 database at
the International Agency for Research on Cancer. Given the function of
p53 in tumor suppression, it is possible that the p53 pathway is
compromised in RCC through other mechanisms, such as the downregulation
of USP10. Indeed, USP10 expression was found to be significantly
decreased in several RCC cell lines including A498, Caki-1, and Caki-2
cells, all of which contain wild-type p53 (FIG. 7A). p53 expression was
also lower in these cells than that of normal renal cells. However, in
RCC cell lines with mutant p53, USP10 levels were increased. USP10 levels
were also decreased in a majority of fresh frozen RCC tissues compared to
corresponding normal tissues (FIG. 7B). The RCC samples with USP10
downregulation all contained wild-type p53 gene (T1-T9), although p53
levels were decreased. These results suggest that downregulation of USP10
might be an alternative way to suppress p53 activity in RCC.
Interestingly, similar as RCC cell lines, USP10 was overexpressed in some
RCC tissues, and these tissues contained mutant p53 (T10, T11). These
results suggest that increased USP10 levels in a mutant p53 background
might be beneficial to tumor growth.

[0084] The expression of USP10 was further examined using RCC tissue
microarray. The staining of USP10 was scored from 0-3, with a score of
0-1 being negative and a score of 2-3 being positive. Representative
staining and scores were shown in FIG. 7c. Strikingly, close to 90% of
clear cell carcinoma exhibited negative staining of USP10. About 50% of
chromophobe and 20% of papillary RCC exhibited negative USP10 staining.
These results suggest that USP10 is downregulated in RCC cases,
especially clear cell carcinoma.

[0085] To confirm the role of USP10 in tumor suppression, USP10 was
reconstituted in RCC cells with USP10 downregulation, and tumor cell
growth was examined using soft agar assay. Reconstitution of USP10 in
CAM-1 and CAKI-2 clear cell carcinoma cell lines, which contain wild-type
p53, restored p53 expression and increased p21 expression (FIG. 8C).
Furthermore, cell proliferation was inhibited with USP10 reconstitution
(FIG. 7D). These results are consistent with the hypothesis that USP10
functions as a tumor suppressor by stabilizing p53.

[0086] USP10 is overexpressed in RCC cell lines and tissues with mutant
p53, correlating with increased p53 levels. This is consistent with a
phenomena that mutant p53 is often overexpressed in many cancers. Since
mutant p53 is often dominant and displays gain of function, increased p53
levels could be advantageous to cancer. In contrast to cells with
wild-type p53, increased expression of USP10 in mutant p53 background
could be beneficial to cancer cell proliferation. Indeed, increased
expression of USP10 in 786-O cells, which contain mutant p53, resulted in
increased cell proliferation, while downregulation of USP10 inhibited
cell proliferation (FIG. 7E and FIG. 8C). These results suggest that
USP10 regulates p53 and cancer cell proliferation in a context-dependent
manner.

[0087] The expression of USP10 in breast and pancreatic cancer cell lines
was examined. As shown in FIG. 16A-B, USP10 was downregulated in a subset
of breast and pancreatic cancer cell lines. In addition, USP10 expression
was lost in many pancreatic cancer tissues (FIG. 16c). These results
further confirm that USP10 might function as a tumor suppressor in
multiple cancers.

[0088] In summary, the results provided herein indicate that in unstressed
cells, USP10 localizes in the cytoplasm and regulates p53 homeostasis.
Following DNA damage, a fraction of USP10 translocalizes to the nucleus
and contributes to p53 activation (FIG. 7F). USP10, through its
regulation of p53, plays a role in tumor suppression.

Example 2

Inhibiting USP10 Polypeptide Activity

[0089] Ubiquitin-AMC (Ub-AMC; BIOMOL), which is a fluorogenic substrate
for a wide range of deubiquitinylating enzymes (Dang et al.,
Biochemistry, 37:1868 (1998)), was used as a substrate of USP10
polypeptides to demonstrate that the deubiquitination of Ub-AMC by USP10
polypeptides is dose dependent. Briefly, the amount of Ub-AMC
deubiquitination in vitro increased as the concentration of USP10
polypeptides increased (FIG. 17A).

[0092] Co-immunoprecipitation assays were performed as follows. Cells were
lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA,
0.5% Nonidet P-40) containing 50 mM β-glycerophosphate, 10 mM NaF,
and 1 mg/mL each of pepstatin A and aprotinin. Whole cell lysates
obtained by centrifugation were incubated with 2 μg of antibody and
protein A or protein G Sepharose beads (Amersham Biosciences) for 2 hours
at 4° C. The immunocomplexes were then washed with NETN buffer
three times and separated by SDS-PAGE. Immunoblotting was performed
following standard procedures.

[0093] Cell growth assays were performed as follows. Cell growth was
analyzed using MTS reagent (Promega) according to the manufacturer's
directions. HCT116 cells stably infected with lentivirus encoding control
shRNA or shRNA designed to reduce USP10 polypeptide expression (1,000
cells/well) were transfected with indicated constructs. After 24 hours,
the cells were plated on 96-well plates and grown on 10% serum containing
media. Cell proliferation was estimated after 1, 2, 3, 4 and 5 days.

[0094] HCT116 cells were harvested and lysed. The resulting cell lysates
were subjected to immunoprecipitation with anti-USP10 polypeptide
antibody and immunoblotted with anti-G3BP1 polypeptide antibodies or
anti-USP10 polypeptide antibodies (FIG. 18). These results demonstrate
that G3BP1 polypeptides interact with USP10 polypeptide in vivo. In
addition, experiments using full length and fragments of full length
USP10 polypeptides indicated that both G3BP1 polypeptides and p53
polypeptides interact with the N-terminal region (e.g., 1-100 amino
acids) of USP10 polypeptides (FIG. 19A).

[0095] In one experiment, HCT116 cells were treated with MG132 for 4 hours
and were depleted of G3BP1 polypeptide expression using shRNA having the
following sequence: 5'-ATGTTTCATTCATTGGAAT-3' (SEQ ID NO:12). MG132 is a
specific, potent, reversible, and cell-permeable proteasome inhibitor. In
another experiment, HCT116 cells transfected with a vector designed to
express a FLAG-G3BP1 polypeptide were treated with MG132 for 4 hours. In
both cases, the cells were lysed, and cell lysates were subjected to
immunoprecipitation with anti-p53 polypeptide antibodies and
immunoblotted with anti-USP10 polypeptide antibodies, anti-p53
polypeptide antibodies, anti-G3BP1 antibodies, and/or anti-FLAG
antibodies.

[0097] In another experiment, HCT116 cells stably transfected with either
a control construct or an shRNA construct designed to reduce USP10
polypeptide expression were transfected with an empty vector, a vector
designed to over-express FLAG-tagged G3BP1 polypeptides, or a vector
designed to over-express FLAG-tagged G3BP2 polypeptides. The shRNA
designed to reduce USP10 polypeptide expression had the following
sequence: 5'-GCCTCTCTTTAGTGGCTCTTT-3' (SEQ ID NO:13). 48 hours later, the
cells were lysed, and cell lysates were blotted with anti-FLAG
antibodies, anti-USP10 polypeptide antibodies, anti-p53 polypeptide
antibodies, or anti-β-actin antibodies. Overexpression of G3BP1
polypeptides, but not G3BP2 polypeptides, decreased the level of p53
polypeptides (FIG. 20). Overexpression of G3BP1 polypeptides did not
change the level of p53 polypeptides in cells depleted of USP10
polypeptides (FIG. 20). These results indicate that G3BP1 polypeptides
regulate p53 polypeptide through USP10 polypeptides.

[0098] In another experiment, HCT116 cells stably expressing either
control construct or an shRNA construct designed to reduce expression of
USP10 polypeptides (USP10shRNA) were transfected with a control vector or
a FLAG-G3BP1 vector. 24 hours later, the cells were plated, and cell
growth was measured by MTS assay at days 1, 2, 3, 4, and 5.
Over-expression of G3BP1 polypeptides significantly enhanced cell growth
in HCT116 cells, but not in HCT116 cells with depleted USP10 polypeptides
(FIG. 21). These results demonstrate that G3BP1 polypeptides regulate
cancer cell growth through USP10 polypeptides.

[0099] In another experiment, HCT116 cells were left untreated or were
treated with 10 Gy irradiation. Two hours later, the cells were lysed.
The resulting cell lysates were subjected to immunoprecipitation with an
anti-USP10 polypeptide antibody and immunoblotted with anti-G3BP1
polypeptide antibodies and anti-USP10 polypeptide antibodies. X-Ray
Irradiation dramatically decreased the interaction between USP10
polypeptides and G3BP1 polypeptides (FIG. 22). These results demonstrate
that DNA damage relieves USP10 polypeptides from G3BP1 polypeptide
inhibition.

Other Embodiments

[0100] It is to be understood that while the invention has been described
in conjunction with the detailed description thereof, the foregoing
description is intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.